WO2017084017A1 - Utilisation de protéine ed-b dans le diagnostic d'une hyperplasie tissulaire - Google Patents

Utilisation de protéine ed-b dans le diagnostic d'une hyperplasie tissulaire Download PDF

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WO2017084017A1
WO2017084017A1 PCT/CN2015/094727 CN2015094727W WO2017084017A1 WO 2017084017 A1 WO2017084017 A1 WO 2017084017A1 CN 2015094727 W CN2015094727 W CN 2015094727W WO 2017084017 A1 WO2017084017 A1 WO 2017084017A1
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protein
cancer
sample
antibody
blood
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Chinese (zh)
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季俊虬
张美�
高美华
陈军
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合肥立方制药股份有限公司
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Priority to PCT/CN2015/094727 priority Critical patent/WO2017084017A1/fr
Priority to US15/776,395 priority patent/US20180340936A1/en
Priority to CN201580084559.XA priority patent/CN108291915B/zh
Priority to EP15908514.1A priority patent/EP3378947B1/fr
Publication of WO2017084017A1 publication Critical patent/WO2017084017A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • the invention relates to a novel method for detecting tissue hyperplasia, belonging to the fields of medicine and biotechnology.
  • the present invention relates to an ED-B domain-containing protein or fragment thereof useful for tumor diagnosis or screening, and the ED-B domain as a marker for tumor and tissue proliferative diseases, which can be used for tumor diagnosis and tumor prognosis evaluation. Early prevention and screening of tumors in the general population.
  • Tumor is the second leading cause of death in non-infectious diseases in the world. Tumor research experts believe that tumors can be prevented, early tumors can be cured, and advanced cancer patients can also improve the quality of life through standardized treatment. However, despite the great advances in modern medical technology, the prognosis for advanced cancer patients, especially when tumors have spread, has a poor prognosis for cancer treatment. In clinical practice, early detection and early treatment of tumors are key, which requires effective diagnostic methods and diagnostic techniques to provide support.
  • Tumor diagnosis methods include: 1. History and physical examination; 2. Endoscopy; 3. Imaging examination such as X-ray examination, ultrasound examination, radionuclide scanning, etc.; 4. Pathological examination, such as cytology examination, living body Tissue examination, etc.; 5, laboratory enzymology or immunological examination. Examination of tumor markers or related substances by laboratory immunology can improve the sensitivity and specificity of early tumor examination, and facilitate the discovery and treatment of tumors. Since some tumor markers are present in the blood of patients, these tumor blood markers provide convenience for early diagnosis of tumors and screening of the general population: small blood collection has good safety and convenience; immunological examination does not require large instruments.
  • the equipment is easy to popularize; the obtained samples can be detected and analyzed in different places; compared with endoscopy and imaging examination, the patient's compliance with immunological examination of blood markers is good. Therefore, the discovery and application of tumor markers or tumor-related substances is an effective method to improve the efficiency of tumor diagnosis.
  • Monoclonal antibodies specific for tumor markers are combined with sensitive immunological detection techniques (ELISA, RIA, IRMA, CLIA, IFA, TRFIA, etc.) to generate a range of methods for tumor marker detection and for clinical practice.
  • ELISA immunological detection
  • RIA RIMARA
  • IRMA IRMA
  • CLIA immunological detection
  • IFA TRFIA
  • the diagnosis and application of tumor markers is more and more widely, such as: tumor screening and screening in the population; tumor diagnosis and confirmation and progress stage judgment; biometric analysis of disease; monitoring of treatment effect and prognosis; determination of secondary tumor and original Tumor; multiple tumor markers Combined application to improve detection efficiency.
  • Tumor markers refer to specific molecules produced by tumor cells or tissues during tumor development. These molecules are low or even difficult to detect in healthy humans, but their content will increase or occur with the occurrence and development of tumors. Significant changes. These molecules may be proteins, polypeptides, nucleic acids, small molecule compounds, and the like. There are nearly 100 kinds of clinically significant tumor markers found at present, and more commonly used CEA, AFP, CYFRA21-1, CA125, CA15-3 and so on. Ideal tumor markers should have high sensitivity and specificity, certain organ specificity, related to tumor growth size or progression, prognosis, and favorable for monitoring tumor recurrence. Only with the above characteristics, far from the ideal level, further research is needed to find.
  • Fibronectin is a multifunctional glycoprotein expressed by epithelial cells, endothelial cells, fibroblasts, hepatocytes, decidual cells, and extravillous trophoblasts.
  • FN soluble in plasma and various body fluids, called plasma fibronectin (pfN), which is involved in blood clotting and other functions; insoluble fibers are present in extracellular matrix and between cells.
  • plasma fibronectin pfN
  • insoluble fibers are present in extracellular matrix and between cells.
  • cFN cell surfaces
  • the application of FN has become increasingly widespread and has been applied to the repair and healing of wounds, sepsis and shock aids, and plays an important role in the pathogenesis of the blood system, cardiovascular and cerebrovascular diseases, and diabetes.
  • the gene of FN is about 75 kb, contains about 50 exons, and has a relative molecular mass of about 250 kDa. It is mainly composed of three kinds of homologous repeating spherical domain units of type I, II and III, and each pair consists of trypsin. Sensitive peptide chain linkage. Each FN subunit has a site of high affinity binding to collagen, cell surface receptors, fibrin, and proteoglycan. Plasma FN is a dimer composed of two similar two subunits, each of which forms two disulfide bonds at the C-terminus, and the entire molecule is V-shaped. Cell FN is a multimer, and the FN subunits of different origins are similar in structure but not identical.
  • Plasma fibronectin (plasma FN, pFN) is 277-513 mg/L in healthy human blood. Its concentration is seen in acute and chronic hepatitis, fatty liver, cirrhosis, obstructive jaundice, cerebrovascular disease, late pregnancy, pancreatic cancer. , lung cancer, cancerous ascites, extensive metastasis of adenocarcinoma; concentration reduction seen in acute leukemia, fulminant hepatic failure, burns, trauma, shock, bacterial or viral infections, acute respiratory distress syndrome, diabetes complicated by ketoacidosis, Uremia, acute circulatory failure, menopausal women, and patients with poor chemotherapy tolerance.
  • FN is an expression product of the same gene, but differs in RNA splicing after transcription, thus producing different mRNAs, producing different FN isoforms, and also differing in post-translational modification. Therefore, in the process of FN gene expression, there are FN(B+) containing ED-B domain (or abbreviated as B-FN or FN (ED-B)) and FN(B-) containing no ED-B domain. Forms. These two forms are thought to play an important role in the development of the individual.
  • ED-B (Extradomain B) is a complete domain comprising 91 amino acids encoded by a single exon in a type III repeat of FN.
  • FN(B+) is rarely expressed in normal adult tissues, but it is increased in the rapidly proliferating tissues such as wound healing tissues and tumor tissues, especially in the process of tumor growth, so it is also called carcinoembryonic gene.
  • FN (B+) containing an ED-B domain is highly expressed in cells such as gastric cancer, colorectal cancer, lung cancer, and breast cancer. Almost all human solid tumors can detect high expression of FN(B+) in both primary and metastatic sites, and FN(B+) may also play an important role in tumor invasion and metastasis.
  • FN(B+) is used as a marker for tumor neovascularization (Carnemolla, Balza et al. (1989). J Cell Biol. 108: 1139-1148.).
  • ED-B protein domain or fibronectin FN (ED-B) containing ED-B domain is not sufficient, and its biological characteristics need further exploration.
  • the invention provides a method of detecting tissue proliferation in a mammal, such as a human, comprising detecting ED-B protein in a biological sample of the mammal, such as a sample of a body fluid, preferably from blood, and in a control sample A comparison of levels of ED-B protein, wherein elevated levels of said ED-B protein in the test sample are indicative of the presence of hyperplasia in said mammal.
  • the method can be used to diagnose the presence or risk of a tumor, to predict a tumor, to diagnose the occurrence or progression of a tumor, to perform a tumor screening, or to monitor the efficacy of a tumor treatment.
  • the invention provides a substance for the specific detection of an ED-B protein in the preparation of a mammal for diagnosis by detecting the level of ED-B protein in a biological sample of a mammal, such as a sample of a body fluid, preferably from blood.
  • a biological sample of a mammal such as a sample of a body fluid, preferably from blood.
  • the invention provides a kit for diagnosing the presence of tissue proliferation in a mammal, the kit comprising a sample for the specific detection of a biological sample of the mammal, such as a body fluid sample, preferably from blood A substance present in the ED-B protein.
  • a biological sample of the mammal such as a body fluid sample, preferably from blood A substance present in the ED-B protein.
  • the kit comprises a conjugate that specifically detects an ED-B protein, preferably the ED-B protein comprises the sequence: (a) SEQ ID NO: 1, or (b) and (a) The sequence in the sequence has a sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more.
  • FIG. 1 SDS-PAGE was used to detect the amount of pED-B in the blood of healthy and tumor patients. From left to right, the four lanes were bovine serum albumin (BSA), ED-B antibody (B5-Fc), tumor pED-B and healthy pED-B; BSA and B5-Fc were controls. The plasma pED-B in tumor patients was significantly higher than that in healthy people.
  • BSA bovine serum albumin
  • B5-Fc ED-B antibody
  • BSA and B5-Fc were controls.
  • the plasma pED-B in tumor patients was significantly higher than that in healthy people.
  • FIG. 2 Western Blot detects the pED-B protein obtained by the pull down method. From left to right, the four lanes were bovine serum albumin (BSA), ED-B antibody (B5-Fc), tumor pED-B and healthy pED-B; the third and fourth lanes were positive for pED-B. And showed B5-Fc positive. The second lane was only positive for B5-Fc (about 55 kD). The first lane was negative.
  • BSA bovine serum albumin
  • B5-Fc ED-B antibody
  • Figure 3 ED-B test kit for the detection of different types of samples.
  • Figure 4A-J Concentrations of pED-B in the blood of healthy and tumor patients as well as other disease patients.
  • Figure 5 Standard curve in the pED-B assay using B5 and L19 antibodies.
  • the marker can serve as a tumor blood marker, providing a basis for screening, diagnosis, treatment, and prognosis of the tumor.
  • the present invention is based on two new findings, a new finding is that blood contains ED-B protein, including part of the plasma fibronectin (pFN) in the blood containing the ED-B domain, or the EF-B in the hydrolyzed fragment of FN Domain, soluble ED-B protein in blood, referred to as pED-B (Plasma ED-B); another new discovery is the level of ED-B protein in blood and various tissue proliferation such as tumor occurrence and malignancy related.
  • pFN plasma fibronectin
  • the invention provides a method of detecting tissue proliferation in a mammal comprising detecting ED-B protein in a biological sample of the mammal, such as a sample of a body fluid, preferably a sample from blood, and ED in a control sample A comparison of the levels of the -B protein, wherein elevated levels of ED-B protein in the test sample are indicative of the presence of tissue hyperplasia in the mammal.
  • the mammal is a human.
  • the tissue proliferation is a solid tumor.
  • level rise means that the measured value obtained by detecting a sample is increased compared to a reference value such as an intermediate value or an average value observed in a patient or a normal person who does not have a tumor, for example, at least 5% increase. At least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times or more.
  • the method of the invention is for use in a method of diagnosing the presence or risk of a disease associated with proliferation, such as a tumor, in a mammal, wherein the ED-B is detected in the sample Elevated protein levels are an indication of the presence or risk of a tumor in the mammal.
  • the methods of the invention are used for prognosis of a proliferative-associated disease, such as a tumor, in a mammal, wherein elevated levels of ED-B protein in the test sample are indicative of poor prognosis in the mammal. .
  • the methods of the invention are used to determine the occurrence or progression of a tumor, for tumor screening or for monitoring the therapeutic efficacy of a tumor, or for individualized administration.
  • ED-B protein refers to a protein comprising an ED-B amino acid sequence, wherein the ED-B amino acid sequence has the amino acid sequence of SEQ ID No. 1 or at least 80% with SEQ ID No. 1, Sequence of sequence identity of 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher.
  • ED-B protein domain or "ED-B domain” as used herein refers to a tertiary structure of a protein formed by the amino acid sequence of SEQ ID No. 1, which is a protein Partially exists.
  • ED-B protein fragment refers to a protein fragment containing an ED-B amino acid sequence formed by denaturation, cleavage, degradation, and hydrolysis of a protein comprising an ED-B domain under physical, chemical, or biological conditions.
  • the ED-B protein is a general term for the above-described proteins, domains and fragments comprising the ED-B amino acid sequence, including but not limited to proteins comprising the ED-B domain or proteins comprising the ED-B peptide.
  • pED-B is an abbreviation for ED-B protein derived from blood, such as plasma.
  • pED-B refers to a protein containing ED-B amino acid sequence in the blood, which can exist in the blood alone, as a domain in the FN or FN hydrolyzed fragment, or in extracellular cells in other blood. Protein-bound forms exist.
  • the pED-B protein is a component in the blood, but may also be a hydrolyzed fragment derived from the cell FN (B+).
  • a synthetic peptide or random mutation based on the amino acid sequence of SEQ ID No. 1 or a protein comprising an ED-B amino acid sequence obtained by genetic recombination also belongs to the ED-B protein of the present invention.
  • the ED-B protein obtained by artificial means is not directly derived from the organism, it is not a pED-B.
  • the protein or protein fragment of the invention may be a phosphorylated or glycosylated protein or a denatured protein or polypeptide fragment.
  • the ED-B protein of the invention has at least 80%, 85%, 90%, 95%, 96%, 97% of the amino acid sequence of SEQ ID No. 1 or with SEQ ID No. 1. a sequence consisting of 98%, 99% or higher sequence identity, or comprising SEQ ID No. 1 or with SEQ ID No. 1 at least 80%, 85%, 90%, 95%, 96%, 97%, Partial sequential ordering of sequences of 98%, 99% or higher sequence identity Column.
  • the ED-B protein or protein fragment of the invention is an ED-B domain-containing FN (B+), and a FN (B+) multimer, dimer, monomer, isomer Body, subtype, hydrolyzed fragment, etc.
  • the invention also relates to a nucleotide sequence encoding an amino acid sequence comprising SEQ ID No. 1 or having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and SEQ ID No. 1. a peptide of 99% or higher sequence identity, or encoded by the amino acid sequence of SEQ ID No. 1 or with SEQ ID No. 1 at least 80%, 85%, 90%, 95%, 96%, 97 A peptide consisting of a sequence of %, 98%, 99% or higher sequence identity.
  • the polynucleotide comprises or consists of the sequence of SEQ ID No. 2.
  • tissue hyperplasia refers to an increase in the number of cells in organs and tissues, resulting in neoplasms, tumors or cancer. Tissue hyperplasia has physiological hyperplasia and pathological hyperplasia. Among them, physiological hyperplasia, such as connective tissue hyperplasia, is an important reaction in the process of wound healing, which plays a positive role in the adaptation of the body. Pathological hyperplasia is mostly caused by excessive stimulation of endocrine hormones or local inflammatory factors, such as breast hyperplasia, benign prostatic hyperplasia, endometrial hyperplasia, intestinal mucosa polyps.
  • polypeptide refers to a polymer of amino acid residues comprising 9 or more amino acids bonded by peptide bonds.
  • the polymer may be linear, branched or cyclic, and may comprise naturally occurring and/or amino acid analogs which may be interrupted by non-amino acids.
  • amino acid polymer is long (e.g., more than 50 amino acid residues), it is preferably referred to as a polypeptide or protein, but if it is 50 amino acids long or shorter, it is preferably referred to as a "peptide.”
  • nucleic acid As used herein, the terms “nucleic acid”, “nucleic acid molecule”, “polynucleotide” and “nucleotide sequence” are used interchangeably to define a polymer of any length, such as polydeoxyribonucleotides (DNA) (for example, cDNA, genomic DNA, plasmids, vectors, viral genomes, isolated DNA, probes, primers and any mixtures thereof) or polyribonucleotide (RNA) molecules (eg mRNA, antisense RNA) or mixed polyribose - Polydeoxyribonucleotides. They contain single or double stranded, linear or circular, natural or synthetic polynucleotides.
  • DNA polydeoxyribonucleotides
  • RNA polyribonucleotide
  • RNA eg mRNA, antisense RNA
  • polynucleotides can include non-naturally occurring nucleotides, such as methylated nucleotides and nucleotide analogs, and can be interrupted by non-nucleotide components. If present, nucleotide modifications can be made before or after polymerization.
  • the ED-B protein of the present invention refers to a protein containing an ED-B amino acid sequence or a proteolytic fragment containing an ED-B amino acid sequence in blood or serum or plasma or contains only one A proteolytic fragment of the ED-B domain.
  • the tissue proliferation of the invention is a tumor.
  • the tumor of the invention is a solid tumor.
  • the solid tumor of the invention may be a benign tumor, a malignant tumor or a borderline tumor.
  • the solid tumor of the invention is an upper gastrointestinal tumor.
  • the upper gastrointestinal tumors of the present invention include, but are not limited to, esophageal cancer, cardiac cancer, laryngeal cancer, and gastric cancer.
  • malignant tumor refers to a tumor or cancer that is often fatal to death, except for the uncontrolled abnormal reproduction of the cells of the tumor itself, as well as the invasion of adjacent normal tissues and the transfer of blood vessels, lymphatic vessels and body cavities to other parts of the body.
  • the tumor of the invention is a malignant tumor, such as a cancer or sarcoma, such as various squamous cell carcinomas, adenocarcinomas, sarcomas.
  • the tumor of the present invention is selected from, for example, esophageal cancer, cardiac cancer, laryngeal cancer, head and neck cancer, gastric cancer, liver cancer, biliary tract cancer, gallbladder cancer, colon cancer, duodenal cancer, lung cancer, bladder cancer, Cervical cancer, ovarian cancer, endometrial cancer, breast cancer, melanoma, pancreatic cancer, kidney cancer, and prostate cancer.
  • the tumor of the invention is an upper gastrointestinal cancer, for example selected from the group consisting of esophageal cancer, cardiac cancer, laryngeal cancer, and gastric cancer.
  • the tumor is nasopharyngeal carcinoma, esophageal cancer, gastric cancer, lung cancer or pancreatic cancer.
  • the biological sample described herein is any biological sample from a test animal, especially any sample comprising a nucleic acid or polypeptide.
  • samples may be included: blood, plasma, platelets, saliva, sputum, urine, and the like, more generally any tissue, organ, or advantageously a biological fluid comprising nucleic acids or polypeptides.
  • Samples for detection in the in vitro methods of the invention should generally be collected in a clinically acceptable manner, for example, in a manner that protects nucleic acids or proteins.
  • the sample to be tested may be a blood sample such as a serum sample and other types of samples.
  • the sample is a blood derived sample, such as a whole blood, serum or plasma sample.
  • the sample can also be pretreated to increase the accessibility of the target molecule, such as by cleavage (mechanical, chemical, enzymatic cleavage, etc.), purification, centrifugation, separation, and the like.
  • the sample can also be labeled to facilitate detection of the presence of the target molecule (fluorescence, radioactivity, luminescence, chemistry, enzymatic labeling, etc.).
  • the biological sample of the invention is a body fluid sample including, but not limited to, blood, saliva, tissue fluid samples, urine, lymph, and cerebrospinal fluid. Liquid samples from various sources can be directly used for testing or pre-treatment after centrifugation, sedimentation, filtration, etc.
  • the biological sample of the invention is a blood sample.
  • the blood sample is a whole blood sample, ie, blood that has not undergone a separation step, which may optionally be diluted.
  • the blood sample of the invention is plasma or Serum samples.
  • the "plasma” as used in the present invention refers to a liquid containing no cell component obtained by centrifugation of whole blood in vitro after anticoagulation treatment.
  • the "serum” as used in the present invention refers to a liquid which is released by blood clots after the whole blood of the whole body is statically coagulated, and the serum does not contain fibrinogen. Since FN is involved in platelet adhesion, aggregation, and thrombus formation, the FN content in serum is generally considered to be lower than plasma.
  • the blood sample of the invention is a plasma sample.
  • the blood sample is sodium citrate as an anticoagulant.
  • the blood sample is collected using a siliconized syringe or a plastic syringe or a silicon coated glass article to avoid activation of the blood coagulation reaction.
  • the blood sample is a fresh or cryopreserved sample.
  • the freshly collected blood sample is directly diluted in 10 volumes of physiological saline or buffer, and the cell-free supernatant is taken as a sample.
  • the relationship between the treatment of various blood samples and/or the content of pED-B contained in the samples obtained after treatment and the content of pED-B in whole blood is well known to those skilled in the art.
  • the ED-B protein of the invention can be detected in a biological sample by any suitable method.
  • the method comprises directly or indirectly determining the level of an ED-B protein of the invention, such as pED-B, in a biological sample, such as a blood sample, such as plasma.
  • the content of the ED-B protein can be determined by a specific binder of the ED-B protein of the present invention, such as a ligand, an aptamer or the like.
  • the specific conjugate is an antibody.
  • the specific conjugate is a nucleic acid aptamer.
  • the content of the ED-B protein can be determined by utilizing physical or chemical characteristics or amino acid sequence characteristics of the ED-B protein of the present invention, for example, by proteomic methods for quantitative analysis of blood.
  • the content of the ED-B protein can be determined by utilizing physical or chemical characteristics or amino acid sequence characteristics of the ED-B protein of the present invention, for example, by proteomic methods for quantitative analysis of blood.
  • the content of the ED-B protein can be determined by utilizing physical or chemical characteristics or amino acid sequence characteristics of the ED-B protein of the present invention, for example, by proteomic methods for quantitative analysis of blood.
  • ED-B egg segments in blood can be detected by immunological techniques including, for example, enzyme-linked immunosorbent assay (ELISA), AlphaLISA, immunoblotting, dot blotting, co-immunoprecipitation, colloidal gold. Immunochromatography, etc.
  • ELISA enzyme-linked immunosorbent assay
  • AlphaLISA AlphaLISA
  • immunoblotting dot blotting
  • co-immunoprecipitation colloidal gold.
  • colloidal gold colloidal gold
  • ELISA enzyme-linked immunosorbent assay.
  • test specimen also referred to as the antigen
  • the capture antibody reacts with the capture antibody on the surface of the solid support to form a capture antibody-antigen complex on the surface of the solid support.
  • an enzyme-labeled detection antibody is added, and the enzyme-labeled detection antibody is bound to the specimen on the surface of the solid phase carrier, and a capture antibody-antigen-enzyme labeling detection antibody diabody is formed at this time.
  • the amount of enzyme on the solid phase is positively correlated with the amount of the specimen in the specimen.
  • a colored product is formed under the catalysis of the enzyme, and the amount of the product is also correlated with the amount of the test substance in the sample, so that qualitative or quantitative analysis can be performed according to the depth of the color.
  • Another application is to form an antibody-antigen complex on the surface of the solid phase carrier, add an unlabeled detection antibody to form a capture antibody-antigen-detection antibody diabody sandwich complex, and then add an enzyme-labeled secondary antibody.
  • a complex of capture antibody-antigen-detection antibody-enzyme-labeled antibody is formed, and subsequent detection is performed by an enzyme-catalyzed substrate.
  • Another application is to immobilize the antigen on the surface of the solid support during the assay, add an enzyme labeling detection antibody to form an antigen-enzyme labeling detection antibody complex, and then complete the detection by an enzyme-catalyzed substrate.
  • Another application is to immobilize the antigen on the surface of the solid phase carrier, add the detection antibody to form an antigen-detection antibody complex, and then add the enzyme-labeled secondary antibody to form an antigen-detection antibody-enzyme-labeled antibody complex. Detection is then completed by enzymatic catalysis of the substrate.
  • the capture antibody is used to capture the antigen to be tested, and the detection antibody is used to detect the total amount of antigen captured by the captured antibody.
  • the detection antibody can be directly labeled with an enzyme, or can be enzymatically labeled with a secondary antibody against the detection antibody.
  • the enzyme label is a product of an enzyme and an antibody or an antigen or a hapten linked by a cross-linking agent, and is a key reagent for ELISA success and failure. It not only has an antibody-antigen-specific immune reaction, but also has an enzymatic reaction, showing the organism. Amplification, but different enzymes use different substrates. Commonly used enzymes for immunological techniques include horseradish peroxidase, alkaline phosphatase, glucose oxidase, and ⁇ -D-galactosidase.
  • the substrates of enzymes commonly used in immunological techniques are o-phenylenediamine (OPD), tetramethylbenzidine (TMB), nitrophenyl phosphate (pNPP), diaminobenzidine (DAB) and the like.
  • OPD o-phenylenediamine
  • TMB tetramethylbenzidine
  • pNPP nitrophenyl phosphate
  • DAB diaminobenzidine
  • the antibodies used in the ELISA may be of monoclonal or polyclonal origin, antibodies may be obtained by immunization of animals, and antibodies may be obtained by genetic engineering techniques and computer-assisted techniques. Preferably, the monoclonal antibody has a higher specificity.
  • the ELISA method can be used to detect liquid samples such as blood, sputum, cell culture fluid, urine, tissue fluid, cerebrospinal fluid, and samples of animal tissues, immobilized tissues, cells, and the like.
  • the animal tissue or cells can be used for ELISA detection after being crushed and homogenized by adding liquid nitrogen grinding or the like.
  • the concentration or amount of the ED-B protein or ED-B protein fragment is detected by a double antibody sandwich enzyme-linked immunosorbent assay.
  • Highly enriched ED-B protein or ED-B protein fragment by capturing ED-B protein or ED-B protein fragment in a biological sample such as a blood sample by an ED-B-specific capture antibody coated on a ELISA plate
  • the antibody-binding of ED-B specific for enzyme labeling is used to improve the sensitivity of detection.
  • FN protein is generally in the blood In the form of a dimer, the "ED-B capture antibody coated on the microplate" and the "anti-ED-B specific detection antibody” may be different for the detection of blood FN by the double antibody sandwich method.
  • the antibody may also be an antibody that recognizes the same antigenic epitope, or may be the same antibody.
  • FN(B+) in blood samples can be detected by double antibody sandwich ELISA.
  • Two antibodies containing FN(B+) can be identified, such as CGS-1 and CGS-2, which can directly or indirectly recognize ED-B. Any two non-competitive antibodies of L19, B5, BC-1, and C6.
  • FN (B+) in blood samples can be detected by a sandwich ELISA method, and an ED-B specific antibody and an FN-specific antibody can also be used.
  • the ED-B protein or ED-B protein fragment or ED-B domain in blood can be detected by the double antibody sandwich ELISA method of the present invention, and can also be used for detecting ED-B protein in tissue homogenate or cell disrupting solution. Fragment. Since FN is a long fibrillar protein with multiple protease hydrolysis sites and is easily broken into multiple small fragments, ED-B or ED-B-containing proteins are detected by two antibodies acting directly on ED-B. Improve the sensitivity and accuracy of detecting ED-B.
  • the ED-B protein or ED-B protein fragment comprises or has at least 80%, 85%, 90%, 95% of the amino acid sequence set forth in SEQ ID NO. Sequence of 96%, 97%, 98%, 99% or higher sequence identity.
  • the ED-B amino acid sequence is encoded by the DNA sequence set forth in SEQ ID NO:2.
  • the ED-B amino acid sequence can be encoded by the DNA sequence set forth in SEQ ID NO:3.
  • the ED-B protein is an FN comprising an ED-B domain.
  • the concentration or amount of the ED-B protein or ED-B protein fragment can also be detected by the AlphaLISA method.
  • AlphaLISA technology is developed based on the principle of interaction of biomolecular substances. For example, an antigen can interact with an antibody, and an antibody can form a microbead complex with a microbead through a molecule interaction, wherein an antibody and a photosensitizer are contained. The beads are combined to form donor beads, and the other antibody is combined with microbeads containing a thiophene derivative reactive with monomeric oxygen to form acceptor beads, which form donor beads in the presence of antigen - Antigen-receptor microbead complex.
  • the laser is used to excite the donor microbeads to release the singlet oxygen molecules, triggering the energy transfer cascade reaction, and the acceptor beads emit light waves, which have strong anti-quenching ability and can be received by the detector. If the biomolecule does not have a specific interaction, the donor microbead-antigen-receptor microbead complex cannot be formed, resulting in no monomer oxygen. When the method spreads to the acceptor beads, there is no light signal.
  • AlphaLISA technology requires the formation of a double antibody sandwich complex as well as the double antibody sandwich ELISA technique, but AlphaLISA has higher sensitivity, uniformity and broader dynamic detection range than ELISA, low sample requirements, simple operation, and no need for washing.
  • the ED-B protein of the present invention can be detected more rapidly, efficiently and sensitively by AlphaLISA.
  • the concentration or amount of the ED-B protein or ED-B protein fragment is detected by the Pull-Down method.
  • the basic principle of Pull-down technology is to immobilize a ligand with affinity to the target protein on a substrate (such as Sepharose).
  • a substrate such as Sepharose.
  • the target protein and the immobilized pro It interacts with the ligand and is adsorbed by the matrix, while the non-target protein flows out with the eluate.
  • the target protein can be recovered by changing the elution conditions.
  • a treated sample such as a blood sample is passed through the resin, and the antibody on the resin captures the ED-B protein in the sample instead of the ED-B protein. Then, the flow-through is carried out, and the non-specific protein on the resin can be further removed by washing or the like.
  • the ED-B protein adsorbed on the resin can be quantitatively determined by ED-B such as pED-B by SDS-PAGE method, Western blot method, or ELISA method, or ultraviolet detection method.
  • the detection of the ED-B protein comprises a protein comprising an ED-B protein domain in a non-specifically concentrated liquid, detected by a method of re-detection of the first enriched protein.
  • the concentration or amount of the ED-B protein or ED-B protein fragment is detected by a dot blot hybridization (Dot blot) method of the protein.
  • Dot blot Dot blot is to place the sample on the membrane, and fix the protein on the membrane or other solid phase materials by drying, baking, ultraviolet irradiation, etc., and then detecting the antibody and the corresponding enzymes and substrates. Perform color development for qualitative or quantitative analysis.
  • the method can enrich a large amount of high-concentration protein on the membrane, and can highly enrich the target protein, which is convenient for detection.
  • the method is shorter in time than the Western blot, and multiple samples can be simultaneously detected on one membrane, and can be used as a gene chip.
  • Western blot can also be used as one of the methods for detecting pED-B
  • electrophoresis methods such as SDS-PAGE can separate proteins with different molecular weights or different charge distributions, and the amount of samples that can be loaded during detection is limited, and pED-B is detected.
  • the protein will reduce the detection sensitivity of pED-B, especially the molecular weight of pED-B protein may be different, and it will show different band distribution when doing Westem blot.
  • the detection method can concentrate the sample by a plurality of samples on the film and dry, which is beneficial to improve the sensitivity of the detection, and can also be sampled by multiple samples-air-dried The operation is performed to further concentrate and concentrate the sample to detect low concentration protein. Dot blots and Western blots of the present invention are well known to those skilled in the art.
  • the concentration or amount of the ED-B protein or ED-B protein fragment is detected by a blood smear method.
  • the procedure is similar to Dot blot. Methods of making blood smears are well known to those skilled in the art.
  • the concentration or amount of the ED-B protein or ED-B protein fragment is detected by colloidal gold immunochromatography.
  • Colloidal gold immunochromatography immobilizes a specific antibody (or antigen) on the membrane in a strip shape, and the colloidal gold labeling reagent (antibody) is adsorbed on the binding pad, and the sample to be inspected is added to the sample pad at one end of the test strip. Moving forward by capillary action, dissolving the colloidal gold labeling reagent on the binding pad and reacting with each other.
  • the combination of the analyte and the gold standard reagent is specifically combined with it. It is trapped and gathered on the test strip, and the color development result can be observed by the naked eye. Based on this method, it has been designed as a diagnostic test strip, which is very convenient to use.
  • the anti-ED-B antibody used such as the antibody pED-B antibody, may be coupled to horseradish peroxidase or to alkaline phosphatase; or by coupling the above peroxidase or The secondary antibody to alkaline phosphatase achieves indirect detection of ED-B such as pED-B.
  • the reaction substrate includes DAB, TMB or a fluorescent substrate and the like.
  • the invention also relates to a method of detecting pED-B. Since pED-B was first discovered in the blood, there are few methods that have been validated for pED-B detection. The Western blot method reported in the literature failed to detect pED-B (Viti, Tarli et al. 1999. Cancer Res.59:347-352.). It is presumed that since the content of pED-B is low, and FN is easily aggregated and precipitated in the blood, it is difficult to detect.
  • the pED-B detection method of the present invention employs the pED-B protein enrichment technique and improves the detection sensitivity of pED-B.
  • One method for enrichment of pED-B is to enrich the pED-B capture on a solid support by a specific method, while the non-pED-B protein can be removed by washing or the like, such as using a specific antibody of pED-B.
  • Capture pED-B; the other is to use a non-specific method to immobilize pED-B and non-pED-B protein mixture on the surface of the solid phase carrier, but to detect the content of pED-B by specific antibodies; pED-B was directly subjected to mass spectrometry.
  • the invention provides a level of detection of a ED-B protein, such as a pED-B protein, in a sample, such as a sample from blood, comprising:
  • an ED-B protein conjugate such as an antibody
  • an ED-B protein conjugate is immobilized on a solid support such as an ELISA plate
  • a second ED-B protein conjugate such as an antibody
  • optionally labeled such as an enzyme (eg, horseradish peroxidase, alkaline phosphatase, glucose oxidase, ⁇ -D-galactosidase) labeling
  • the second ED-B protein conjugate binds to a different epitope on the ED-B protein with the first ED-B protein conjugate;
  • an antibody against the second ED-B protein conjugate optionally labeled, such as an enzyme (eg horseradish peroxidase, alkaline phosphatase, glucose oxidase, beta-D-half) Galactosidase), as well as
  • an enzyme eg horseradish peroxidase, alkaline phosphatase, glucose oxidase, beta-D-half
  • Galactosidase eg. horseradish peroxidase, alkaline phosphatase, glucose oxidase, beta-D-half
  • the detection can be a color reaction known in the art, for example using the substrate o-phenylenediamine (OPD), tetramethylbenzidine (TMB), nitrophenyl phosphate (pNPP), Diaminobenzidine (DAB) and the like.
  • the first and second ED-B protein conjugates are ED-B protein specific binders, for example independently selected from B5 and L19.
  • the invention provides a substance for specifically detecting an ED-B protein for detecting a level of ED-B protein in a biological sample of a mammal such as a human, such as a sample of a body fluid, preferably a sample from blood, for diagnosing tissue in a mammal Proliferation, the presence or risk of a solid tumor, the prognosis of a solid tumor, the determination of the occurrence or progression of a solid tumor, the screening of a solid tumor, or the monitoring of the efficacy of a solid tumor.
  • the mammal is a human.
  • the invention provides a substance for the specific detection of ED-B protein in the preparation of a diagnostic for the level of ED-B protein in a sample of a biological sample, such as a body fluid, preferably from blood, in a mammal, such as a human.
  • tissue hyperplasia in mammals, the presence or risk of a solid tumor, the prognosis of a solid tumor, the determination of the occurrence or progression of a solid tumor, the screening of a solid tumor, or the monitoring of the efficacy of a solid tumor treatment.
  • the mammal is a human.
  • the invention provides for diagnosing tissue proliferation in a mammal, the presence or risk of a solid tumor, prognosis of a solid tumor, determining the occurrence or progression of a solid tumor, performing a solid tumor screening or monitoring a solid tumor
  • a kit for the therapeutic efficacy comprising a substance for specifically detecting a biological sample of the mammal, such as a body fluid sample, preferably a sample from blood, in an ED-B protein.
  • the breastfeeding Animals are people.
  • the substance specifically detecting the ED-B protein of the present invention is a specific conjugate of an ED-B protein such as pED-B by detecting a biological sample such as a body fluid sample, preferably in blood, plasma or serum.
  • ED-B protein such as pED-B level, assists in determining the occurrence or progression of a tumor, or screening a high-risk population for tumor screening, or judging the prognosis of a tumor patient, or determining whether treatment for a tumor patient is effective, or for Individualized medication.
  • Auxiliary diagnosis refers to the use of serology, imaging, pathology and other means to assist physicians in judging the type and severity of the disease and improving the accuracy of the diagnosis.
  • Prognosis refers to predicting the likely course and outcome of a disease. It includes determining the specific consequences of a disease and predicting the likelihood that a certain outcome will occur within a certain period of time.
  • Individualized medication means that the treatment is based on the characteristics of each patient, such as genetic factors, gender, age, weight, physiology, pathological characteristics and other drugs being taken, based on a comprehensive situation, which is safe, reasonable, effective and economical. Drug treatment program.
  • the "specific conjugate of ED-B protein” as used in the present invention refers to a molecule capable of recognizing and binding to the ED-B protein or ED-B protein fragment of the present invention, which molecule is ED-B according to the present invention.
  • the protein or ED-B protein fragment has specificity and high affinity, and also includes molecules that recognize the ED-B protein or fragment of the cell surface or extracellular matrix with high affinity and specificity.
  • the specific binder can be a protein or a nucleic acid.
  • the specific conjugate of the ED-B protein of the invention is an antibody specific for an ED-B protein, such as pED-B.
  • the antibody is a polyclonal antibody or a monoclonal antibody or an antigen-binding fragment of an antibody, eg, a polypeptide comprising an antibody variable or variable region sequence, scFv, Fab, Fab', F(ab') 2 .
  • the antibody of the invention may be an antibody obtained by immunizing an animal, such as an immunized mouse, rat or rabbit.
  • the antibodies of the invention can be obtained by computer simulation design or artificially assisted design based on antibodies obtained by immunization or by genetic synthesis.
  • the ED-B domain is highly conserved among different species, the ED-B amino acid sequences of human, mouse, rat, dog and rabbit are completely identical, and are also highly homologous to chicken ED-B.
  • the ED-B of the present invention is highly homologous in mammals and therefore has similar biological characteristics in different species, and antibodies specific for directly recognizing ED-B surface antigen generally do not have species specificity, such as CGS- 1.
  • CGS-2 PCT/GB97/01412; Nissim, Hoogenboom et al. (1994). EMBO J. 13: 692-698), L19 (Pini, Viti et al. (1998). J Biol Chem.
  • Antibodies that indirectly recognize ED-B may be species-specific, such as BC-1 (Carnemolla, Balza et al. (1989). J Cell Biol. 108: 1139-1148.), C6 (Ventura, Sassi et al). (2010). PLoS One.5: e9145.) and other antibodies that recognize epitopes on human ED-B adjacent domains.
  • kits of the invention can be used to detect the content of a protein fragment of the invention in a biological sample, such as a body fluid sample, preferably a sample from blood.
  • the kit of the invention comprises an antibody to at least two ED-B proteins.
  • Kits of the invention include ED-B specific antibodies, such as L19 and/or B5.
  • the L19 and B5 antibodies are capture antibodies and enzyme-labeled antibodies, respectively, for detection of ED-B protein or ED-B protein fragments. Binding of either antibody to ED-B did not affect the binding of the other antibody to ED-B, suggesting that the epitopes for binding of L19 and B5 antibodies to ED-B are not identical. Since FN(B+) also has the biological characteristics of forming dimers, even the use of antibodies recognizing the same epitope can be used for kit development.
  • a composition or kit of the invention can be used to high risk by detecting the level of an ED-B protein, such as pED-B, of the invention in a biological sample, such as a body fluid sample, preferably a sample from blood.
  • a biological sample such as a body fluid sample, preferably a sample from blood.
  • the population is screened for tumors and the likelihood of developing a tumor in the sample provider is determined by measuring the ED-B protein, such as pED-B content, in a sample of a suspected tumor patient, in comparison with a healthy control.
  • the method can also be used to determine if an individual has a tumor.
  • a composition or kit of the invention can be used to determine the prognosis of a tumor patient by detecting the level of an ED-B protein, such as pED-B, in a biological sample, such as a body fluid sample, preferably a sample from blood.
  • the prognosis of the sample provider is determined by measuring the ED-B protein, such as pED-B content in a tumor patient sample, against the ED-B protein, such as pED-B content, in a healthy control or a patient's previous corresponding sample.
  • ED-B protein such as pED-B content
  • a composition or kit of the invention can be used to determine a tumor by detecting a level of an ED-B protein, such as pED-B, of the invention in a biological sample, such as a sample of a body fluid, preferably a sample from blood.
  • a biological sample such as a sample of a body fluid, preferably a sample from blood.
  • the patient's therapeutic effect by measuring tumor patients
  • the ED-B protein in the sample such as the pED-B content
  • the level of ED-B protein, such as pED-B is used to assist in determining whether the treatment maintains or alters the therapeutic strategy.
  • compositions or kits of the invention can be used to individualize by detecting levels of the protein ED-B, such as pED-B, of the invention in a biological sample, such as a sample of a body fluid, preferably a sample from blood.
  • a biological sample such as a sample of a body fluid, preferably a sample from blood.
  • the therapeutic effect of the sample provider is judged by comparing the ED-B protein in the sample of the tumor patient, such as the pED-B content, with the ED-B protein, such as the pED-B content in the healthy control or the patient's previous corresponding sample.
  • Individualized treatment is performed based on the level of ED-B protein, such as pED-B, to assist in determining whether the treatment maintains or alters the treatment strategy.
  • the tumor is a solid tumor.
  • Solid tumors can be benign tumors, malignant tumors, and junctional tumors.
  • a malignant tumor can be a cancer or a sarcoma, such as various squamous cell carcinomas, adenocarcinomas, and sarcomas.
  • the tumor is selected from the group consisting of esophageal cancer, nasopharyngeal carcinoma, cardiac cancer, laryngeal cancer, head and neck cancer, gastric cancer, liver cancer, biliary tract cancer, gallbladder cancer, colon cancer, duodenal cancer, lung cancer, bladder cancer , cervical cancer, ovarian cancer, endometrial cancer, breast cancer, melanoma, pancreatic cancer, kidney cancer and prostate cancer.
  • the tumor is an upper gastrointestinal cancer, for example selected from the group consisting of esophageal cancer, cardiac cancer, laryngeal cancer, and gastric cancer.
  • the tumor is nasopharyngeal carcinoma, esophageal cancer, gastric cancer, lung cancer or pancreatic cancer.
  • the kit of the present invention comprises a monoclonal antibody B5 or L19 antibody which specifically recognizes the ED-B domain of FN and has high affinity and high sensitivity.
  • the present invention also develops an ELISA test kit based on the detection method of pED-B in blood.
  • ELISA is a commonly used method for protein immunoassay.
  • the kit of the invention includes both B5 and L19 antibodies as candidate antibodies for double antibody sandwich ELISA.
  • the kit of the present invention further comprises an anticoagulant capable of preventing blood coagulation, such as natural anticoagulant heparin, hirudin, etc., divalent calcium ion chelating agent sodium citrate, ethylenediaminetetraacetic acid (EDTA), etc.
  • an anticoagulant capable of preventing blood coagulation, such as natural anticoagulant heparin, hirudin, etc., divalent calcium ion chelating agent sodium citrate, ethylenediaminetetraacetic acid (EDTA), etc.
  • the blood product or plasma treated with the anticoagulant can be used for the detection of the ED-B detection method described in the present invention.
  • the invention is used in the acquisition of plasma Sodium citrate is used as an anticoagulant to reduce the precipitation loss of FN.
  • the present invention finds that the ED-B protein is relatively stable in a healthy human body, and is greatly increased in the blood of tumor patients and tissue hyperplasia patients, it suggests that pED-B is closely related to the occurrence and development of tumors. Therefore, the ED-B protein of the present invention can be used as a tissue hyperplasia and tumor marker. pED-B can be widely used as a tumor blood marker in medical diagnosis, treatment and scientific research.
  • the level of the marker in the blood of the present invention it is helpful to determine whether there is a tumor in the body, for use in screening a high-risk population for tumor screening, or to help judge the progress of the tumor, or to help To judge the prognosis of tumor treatment, or to help individualized medication and precision treatment, or for biological research of tumors.
  • A) Construction of serum marker standard vectors The DNA sequence shown in SEQ ID NO: 3 can be synthesized, and the DNA sequence can encode three domains of FN (NB7), and the encoded amino acid sequence is described in the literature (Schiefner, Gebauer et al. (2012). J Biol Chem. 287:17578-17588.).
  • FN (7B8) the DNA sequence also encodes a signal peptide at the N-terminus of FN (7B8) and 6 histidine tags at the C-terminus, wherein the signal peptide facilitates protein secretion expression of FN in mammalian cells, group The amino acid tag facilitates protein purification.
  • N-terminus and C-terminus of this sequence were cloned into the pCI-neo vector (Promega Corporation) by Nhe I and Not I restriction endonuclease recognition sites, respectively, to form a pCI-neo-7B8 vector.
  • B Protein expression of B5, L19 antibodies.
  • the DNA sequence of the single-chain antibody fragment (scFv) structure of B5 and L19 was synthesized by gene (see Patent Application No. CN201480001324.5, Pini, Viti et al. (1998). J Biol Chem. 273: 21769-21776 and Borsi, Balza et al. (2002). Int J Cancer. 102: 75-85).
  • a DNA sequence encoding a signal peptide is added to the 5' end of the DNA sequence of the antibody and a DNA sequence encoding an IgG1 Fc tag is added at the 3' end, wherein the signal peptide is used for protein secretion expression in mammalian cells, and IgG1 Fc is advantageous for antibodies.
  • the N-terminus and C-terminus of this sequence were digested with Nhe I and Not I restriction endonucleases, respectively, and cloned into pCI-neo vector to form pCI-neo-B5-Fc vector and pCI-neo-L19-Fc vector.
  • the antibodies described in this example all adopt the form of a single-chain antibody, and the antibody Fc fragment contains a hinge region, and the obtained antibody has a molecular weight of about 52 kD.
  • the antibodies described in the examples of the invention generally all contain an Fc protein structure. Vector construction and expression patterns of antibody proteins are commonly used in the art (Borsi, Balza et al. (2002). Int J Cancer. 102: 75-85.).
  • the obtained antibody-containing fusion protein-containing plasmid was extracted in large amounts, and the liposome reagent of Invitrogen was used.
  • the plasmid was transfected into CHO-K1 cells in 2000, and the neo gene of the pCI-neo plasmid vector was used in a selective medium containing G418 (Davies and Jimenez (1980). Am J Trop Med Hyg. 29: 1089-1092.). After selective culture for 4 weeks, the cloned culture was carried out by the limiting dilution method, and the obtained monoclonal cells were further cloned and cultured to obtain a stable strain.
  • the obtained cell line was expressed in suspension with Hyclone CD4 CHO.
  • the FN (7B8) protein was affinity purified using a nickel column, and the Fc-tagged antibody protein was purified using Protein A.
  • the purified protein was purified by SDS-PAGE and the purity of the sample was obtained. The concentration of the sample was measured by an ultraviolet spectrophotometer, and the binding ability of FN (7B8) to the antibody was evaluated by ELISA.
  • Example 2 Detection of FN (B+) in plasma using L19-IL2 antibody fusion protein and B5-Fc antibody (sandwich ELISA)
  • FN(B+) in blood samples can be detected by sandwich ELISA.
  • Two antibodies that recognize FN(B+) can be used, such as CGS-1, CGS-2, L19, which can directly or indirectly recognize ED-B. Any two non-competitive antibodies in B5.
  • FN (B+) in blood samples can be detected by sandwich ELISA, and an FN-specific antibody and a ED-B specific antibody.
  • L19-IL2 5 parallel samples were coated on the plate.
  • the capture antibody L19-IL2 was diluted with a carbonate buffer (Na 2 CO 3 1.59 g/L, NaHCO 3 2.93 g/L) at a concentration of 9.6 to a concentration of 2 ng/ ⁇ l, and coated with an enzyme plate at 100 ⁇ l/well. .
  • the plate was placed in a 37 ° C biochemical incubator for 2 h.
  • the reaction was terminated: the reaction was terminated with 2 mol/L of H 2 SO 4 , and 100 ⁇ l/well of the stop solution was added to the sample well to terminate the reaction, and the color of the sample well changed from blue to yellow immediately.
  • Double-wavelength detection of OD450nm and OD630nm was selected using a microplate reader. After subtracting the background value of the blank control well, the sample is tested for absorbance.
  • the absorbance value obtained by the indirect method of enzyme-labeled mouse anti-human antibody was judged.
  • the coloration value of B5 and L19 combination was significantly higher than that of the negative control, that is, the sensitivity of the combination of B5 and L19 antibody satisfies the concentration detection requirement of pED-B. Results are shown in Figure 5.
  • Healthy balb/c nu nude mice were housed in a SPF-class barrier system to 20 g/head, a total of 12 rats, of which 6 in the experimental group were implanted subcutaneously into the human pharyngeal squamous cell carcinoma FaDu (Rangan (1972). Cancer. 29: 117-121.) About 2 million cells, continue to raise. When the tumor diameter of the mouse grows to an average of 2 cm (about 4 weeks), the whole blood of the mouse is taken, and sodium citrate is used as an anticoagulant, and the mixture is centrifuged at 3000 g for 2 minutes at 4 ° C, and the plasma supernatant is discarded and repeated once. The ELISA method was used to detect the content of ED-B in plasma.
  • the plasma samples of 6 healthy balb/c nu mice were used as control samples to detect the content of FN(B+) in the blood of mice.
  • a method for detecting the content of FN(B+) in the blood of mice in general, was coated with a CO19 96-well microtiter plate coated with a L19-Fc antibody (2 ng/ ⁇ l, 100 ⁇ l/well) at 37 ° C for 2 hours, using BSA in PBS. The solution was blocked overnight at 4 ° C, washed 3 times with PBS, 100 ⁇ l of mouse plasma diluted 10 times with PBS was added, incubated for 2 hours at room temperature, and washed thoroughly, then HRP-labeled B5-Fc antibody (2 ng / ⁇ l, 100 ⁇ l / well) was added. , using TMB method to develop color.
  • the ED-B content in the blood of tumor-bearing mice was 0.66 ⁇ 0.25 mg/L. Compared with healthy mice, the plasma ED-B content in tumor-bearing mice increased.
  • Example 4 Human plasma sample collection, pull-down method for determination of pED-B and tumor malignancy
  • the blood of healthy people and the blood of the tumor patients were treated with sodium citrate anticoagulant, and then centrifuged at 4 ° C, 3000 g for 2 minutes, and the plasma was taken, and the precipitate was discarded and repeated once.
  • Protein A (Genscript Co.) filler 50 ⁇ l of Protein A (Genscript Co.) filler was pretreated with Binding wash buffer, transferred into a 1.5 ml centrifuge tube, 0.2 mg of B5-Fc antibody was added, and mixed for 5 hr at 4 ° C to form Protein A and B5-Fc antibody II. Poly complex. The supernatant was removed by centrifugation, and Protein A was washed three times with Binding wash buffer, the supernatant washing solution was discarded, and the Protein A and B5-Fc antibody dimeric complexes were equally divided into 3 portions, and then added to three samples, respectively.
  • 100 ⁇ l of healthy plasma B) 100 ⁇ l of esophageal cancer patient plasma, C) BSA solution, negative control. The mixture was mixed by inversion at 4 ° C for 1 hr and washed three times with PBS. The complex precipitate was then taken for sample processing for SDS-PAGE detection.
  • the concentration of the separation gel used in SDS-PAGE was 8%, the concentration of the concentrated gel was 5%, the volume of the sample was 20 ⁇ L, the current was 30 mA, and Coomassie blue staining was performed. According to the results of SDS-PAGE analysis, B5-Fc-captured proteins were present in the plasma of healthy human plasma and selected tumor patients, and the protein concentration captured from the plasma samples of tumor patients was significantly higher than that of healthy human plasma. see picture 1.
  • Example 5 Determination of ED-B content in blood and tumorigenesis
  • the ELISA method described in Example 2 was used to detect healthy people and newly diagnosed tumor patients (including nasopharyngeal carcinoma, esophageal cancer, gastric cancer, lung cancer, pancreatic cancer, leukemia, lymphoma patients, confirmed by pathology or imaging diagnosis), benign Plasma ED-B levels in patients with tissue hyperplasia (including mammary gland hyperplasia, benign tumors) and some chronic diseases (including hyperlipidemia, rheumatoid, pneumonia) (see Figure 4A-J).
  • tumor patients including nasopharyngeal carcinoma, esophageal cancer, gastric cancer, lung cancer, pancreatic cancer, leukemia, lymphoma patients, confirmed by pathology or imaging diagnosis
  • benign Plasma ED-B levels in patients with tissue hyperplasia including mammary gland hyperplasia, benign tumors
  • some chronic diseases including hyperlipidemia, rheumatoid, pneumonia
  • the levels of pED-B in the blood of esophageal cancer, gastric cancer, nasopharyngeal carcinoma, and lung cancer patients are significantly increased, and the difference is statistically significant compared with healthy people.
  • the levels of pED-B in the blood of patients with benign tumors and hyperplasia of mammary glands also increased, and the difference was statistically significant compared with healthy people.
  • the statistical method is the Mann-Whitney test.
  • ED-B enzyme-linked immunosorbent kit consisting of: an enzyme-labeled plate coated with L19-Fc antibody; horseradish peroxidase (HRP)-labeled B5-Fc antibody; ED-B standard; sample dilution Liquid; washing liquid; color developing agent;
  • HRP horseradish peroxidase
  • the plate was placed in a 37 ° C biochemical incubator for 2 h, placed in a 4 ° C refrigerator, and incubated for 8-10 h.
  • HRP horseradish peroxidase
  • B5-Fc antibody The sugar molecule on the surface of HRP is oxidized to an aldehyde group by NaIO 4 , and then HRP is combined with the amino group on the antibody, and the enzyme-labeled antibody obtained by the method The yield is high and there is no significant loss of enzyme and antibody activity (Tsang, Greene et al. (1995). J Immunoassay. 16: 395-418.).
  • PBST 1 L, that is, Tween-20 was added to the PBS in a volume ratio of 0.5 Torr, and mixed.
  • the substrate color developing solution includes the A liquid and the B liquid, and the A liquid and the B liquid are mixed in an equal volume before use, and the color is developed in the dark.
  • liquid A 1 g of urea peroxide, 35.8 g of disodium hydrogen phosphate, 10.2 g of citric acid and ddH 2 O are dissolved in the dark, and then added to Tween-20100 ⁇ l, and the volume is adjusted to 1 L, and stored at 4 ° C in the dark;
  • B liquid The preparation process of B liquid is: TMB 0.7g, citric acid 10.3g, dissolved in 40ml of DMSO, protected with light, and then added to ddH 2 O to 1L, and stored at 4°C in the dark.
  • Stop Solution 98% concentrated sulfuric acid and ultrapure water were mixed at a volume ratio of 1:8 to obtain a 2 mol/L sulfuric acid solution.
  • Example 7 ELISA kit of the invention detects ED-B content in various samples
  • Serum acquisition fresh blood of 20-week-old Bal B/C mice was collected and placed in a 1.5 mL centrifuge tube, tilted or placed flat in a 37 ° C biochemical incubator for 1 h. After the serum was precipitated, centrifuge (3000 g, 5 min) 2 Then, pipette the supernatant, taking care not to suck red blood cells. The supernatant was taken for ELISA detection. The sample to be tested is placed on ice or in a refrigerator at 4 ° C for a short period of time and stored at -20 ° C or lower for long-term storage.
  • Plasma acquisition When collecting 20-week old Bal B/C mice fresh blood, it is necessary to add appropriate amount of sodium citrate anticoagulant, and place the blood collection tube vertically for 1 hour to precipitate the blood naturally, then centrifuge with a centrifuge tube and centrifuge (3000g). 2 times), separated plasma for detection.
  • the sample to be tested is placed on ice or in a refrigerator at 4 ° C for a short period of time and stored at -20 ° C or lower for long-term storage.
  • FaDu cultured FaDu cells were scraped and transferred into a centrifuge tube.
  • the suspension cells were directly transferred to a centrifuge tube, centrifuged at 1000 g for 5 min, washed with PBS for 3 times, discarded with PBS, added with liquid nitrogen and ground with a grinding rod, and then added.
  • the sample to be tested is placed on ice or in a refrigerator at 4 ° C for a short period of time and stored at -20 ° C or lower for long-term storage. Total protein concentration was determined by the BCA method.
  • Tissue sample processing freshly collected human pharyngeal squamous cell carcinoma (FaDu) Bal b/c nude mice, wash away blood stains, add protease inhibitors as needed, cut the animal tissue and add liquid nitrogen. It is ground in a mortar to form a fine powder. Add appropriate amount of PBS, centrifuge (3000g, 5min) twice, and take the supernatant for ELISA. The sample to be tested is placed on ice for a short time or in a refrigerator at 4 ° C. It should be stored at -20 ° C or -80 ° C for a long time. Total protein concentration was determined by the BCA method.
  • Termination of the reaction The reaction was terminated with 2 M H 2 SO 4 , and 100 ⁇ l/well of the stop solution was added to the sample to terminate the reaction, and the sample empty color immediately changed from blue to yellow.
  • the ELISA of the present invention provides a method for quantitatively determining ED-B content
  • the detectable samples are: blood samples, including serum, plasma, fresh blood diluted samples, and cell and tissue samples. Etc.
  • the results are shown in Figure 3.

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Abstract

La présente invention concerne l'utilisation d'une protéine ED-B en tant que marqueur d'une hyperplasie tissulaire (telle qu'une tumeur), un procédé de détection de la protéine ED-B, ainsi qu'une trousse de diagnostic comprenant la protéine ED-B. Ladite trousse comprend un anticorps génétiquement modifié dirigé contre l'ED-B marqueur tumoral.
PCT/CN2015/094727 2015-11-16 2015-11-16 Utilisation de protéine ed-b dans le diagnostic d'une hyperplasie tissulaire WO2017084017A1 (fr)

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PCT/CN2015/094727 WO2017084017A1 (fr) 2015-11-16 2015-11-16 Utilisation de protéine ed-b dans le diagnostic d'une hyperplasie tissulaire
US15/776,395 US20180340936A1 (en) 2015-11-16 2015-11-16 Use of ed-b protein in diagnosis of tissue hyperplasia
CN201580084559.XA CN108291915B (zh) 2015-11-16 2015-11-16 Ed-b蛋白在诊断组织增生中的应用
EP15908514.1A EP3378947B1 (fr) 2015-11-16 2015-11-16 Utilisation de protéine ed-b dans le diagnostic d'une hyperplasie tissulaire

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